Yeast Cell Cycle FACS protocol
GDMC: YEAST CELL CYCLE BY FLOW CYTOMETRY Modified from Dan Gottschling S. cerevisiae protocol with added detail from Susan Forsburg S. Pombe protocol. YOU WILL NEED TO HAVE BEFORE: 1. Cold 70% ethanol made from sterile water and 100% ethanol GDMC SOURCE AND PART NUMBER TO ORDER THIS: 2. 0.5M Na Citrate stock GDMC SOURCE AND PART NUMBER TO ORDER THIS: 3. 10 mg /ml RNAse A stock (Boil for 10 mins, cool, filter, and store at -20C) GDMC SOURCE AND PART NUMBER TO ORDER THIS: 4. 1 mg/ml Propidium Iodide stock (filter and store in the dark at -20C) GDMC SOURCE AND PART NUMBER TO ORDER THIS: DAY 0: 1. Make sure that you have all of the stocks and solutions that you need. You should also have control strains for 1X, 2X, and 4X DNA prepared (see “Preparing Control Strains” at the end). 2. Start an overnight culture of your cells in 3ml YPD liquid in the 30C shaker . ALTERNATIVE OPTION: See “Seeding for log growth in one step” at the end. DAY 1: 1. Seed two new 3ml YPD liquid cultures of your strain, for each strain, first thing in the morning. 2. In 4-8 hours, measure the OD600 of each culture by taking 100ul from your culture, and adding it to 900ul of YPD liquid in a cuvette. Use fresh YPD as a blank. This will give you the number of cells / ml in the 1:10 dilution; multiply this by 10 to get the number of cells per ml in your culture. 3. Put 70% ethanol on ice to cool. Measure out the amount from your culture that will have 1E7 cells and centrifuge at 2000 rpm for 5 minutes. Remove the supernatant. 4. Add 1ml of ice-cold 70% ethanol to your tube while vortexing, to fix the cells. These cells can now be stored at 4C forever to continue later or use again if you want. 5. Using your 0.5M Na Citrate stock and 10mg/ml RNAse A stock, create some 50mM Na Citrate solution, AND some 50mM Na Citrate / 0.25 mg/ml RNAse A solution. EXAMPLE DILUTIONS USING STOCK CONCENTRATIONS ABOVE: 6. When you are ready to process your cells, take 0.3ml from your cells you made in step 4, and add them to 3ml of 50 mM Na Citrate, in a 5ml sterile tube. Vortex to mix, and spin at 2000 rpm for 5 minutes. 7. Remove supernatant, and resuspend the pellet in 0.5ml of 50mM Na Citrate, 0.25 ug/ml RNAse A. Put in a shaker or rotator at 50C for one hour, or ALTERNATIVELY put at 37C overnight. The DAY # will assume you used 37C overnight. DAY 2: 1. Pellet cells (2000 rpm, 5 min) and wash in 1ml of 50mM Na Citrate. Pellet again (2000 rpm, 5 min) and resuspend in 1ml of 50mM Na Citrate. 2. Add 16μl of 1mg/ml Propidium Iodide, for final concentration of 16 μg/ml PI. OPTIONAL: Sonicate your cells for 45s just before FACS to separate clumped cells if this is a problem. APPENDIX 1: SEEDING FOR LOG GROWTH IN ONE STEP To avoid having to start a new culture in the morning and wait for 4-8 hours to get log phase growth, you can start a series of dilutions overnight and one of them will easily be in log phase. 1. For each strain, set up 6 tubes with 3ml YPD per tube. 2. Seed tube #1 just as you normally would for overnight culture. 3. Mix well, then transfer 400ul from tube #1 to tube #2. Repeat for tube #3, 4, etc. The next day, one of your tubes will be in log phase of growth when you arrive in lab, and you can immediately begin processing for FACS with DAY 1 step 2. APPENDIX 2: Preparing Control Strains You will need control cells for 1X, 2X, and 4X DNA content. Prepare these from starved haploid cells (1X), dividing haploid cells (2X), and dividing diploid cells (4X). Day 0: 1. Seed overnights of wild-type haploid and wild-type diploid to overnight liquid YPD cultures at 30C. Make TWO tubes of haploid cells. Day 1: 1. Seed haploid and diploid cells to new YPD liquid cultures, wait 4-8 hours for good log phase growth. Fix these cells in 70% ethanol (Day 1 steps 2,3,4 of main protocol above). These cells can be stored at 4C forever and used as peak controls in FACS cell cycle experiments. The haploid for 2X, the diploid for 4X. 2. Spin down the second tube of haploid cells for 5 min at 2000 rpm. Resuspend in 3ml of SLSM (See GNA Sporulation protocol on the wiki for SLSM recipe), and shake overnight at 25C. Day 2: 1. Fix the cells from SLSM overnight in 70% ethanol (Day 1 steps 2,3,4 of main protocol above). These cells can be stored at 4C forever and used as peak controls in FACS cell cycle experiments, for 1X. REFERENCES http://labs.fhcrc.org/gottschling/Yeast%20Protocols/facs.html http://pingu.salk.edu/flow/protocols/ycc.html